Bottom Line:
Primary human skeletal muscle cells have recently become available from a number of commercial vendors.However, only limited characterization of these cells has been reported to date.Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification.

ABSTRACTThere is a significant unmet need for safe, anabolic muscle therapies to treat diseases and conditions associated with severe muscle weakness and frailty. The identification of such therapies requires appropriate cell-based screening assays to select compounds for further development using animal models. Primary human skeletal muscle cells have recently become available from a number of commercial vendors. Such cells may be valuable for studying the mechanisms that direct muscle differentiation, and for identifying and characterizing novel therapeutic approaches for the treatment of age- and injury-induced muscle disorders. However, only limited characterization of these cells has been reported to date. Therefore, we have examined four primary human muscle cell preparations from three different vendors for their capacity to differentiate into multinucleated myotubes. Two of the preparations demonstrated robust myotube formation and expressed characteristic markers of muscle differentiation. Furthermore, these myotubes could be induced to undergo morphological atrophy- and hypertrophy-like responses, and atrophy could be blocked with an inhibitor of myostatin signaling, a pathway that is known to negatively regulate muscle mass. Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification. Taken together, our results indicate that primary human muscle cells can be a useful system for studying muscle differentiation, and may also provide tools for studying new therapeutic molecules for the treatment of muscle disease.

Fig2: Induction of atrophy and hypertrophy in HSMM and SkMDC myotubes. (A) Differentiated myotubes were treated with a concentration series of Mstn or Dex for 48 h. Representative images of MYH2/Hoechst-labeled myotubes treated with 1 μg/ml Mstn or 50 μM Dex for 48 h. Plots of the percent decrease, from untreated cells, in myotube area at each concentration of test agent are shown. (All plots represent means ± SEM, n = 15. *p < 0.05 vs. untreated using Student’s t test. All images at ×10 magnification; scale bar = 100 microns). (B) Differentiated myotubes were treated with a concentration series of IGF-1 for 48 h. Representative images of MYH2/Hoechst-labeled myotubes following treatment with 1 μg/ml insulin-like growth factor-1 (IGF-1). Plots show the percent increase, from untreated cultures, in myotube area at each concentration of IGF-1. (All plots represent means ± SEM, n = 15. *p < 0.05 vs. untreated using Student’s t test. All images at ×10 magnification; scale bar = 100 microns). (C) Western blots of myotube lysates from cultures treated with 1 μg/ml Mstn, 50 μM Dex, or 1 μg/ml IGF-1 for 48 h and probed with antibodies to phosphorylated- and total Smad2 and phosphorylated- and total AKT. Anti-alpha tubulin was used as a loading control.

Mentions:
Dexamethasone, a synthetic steroid, and myostatin, a growth factor which is a negative regulator of muscle mass, are well-characterized inducers of myotube atrophy, as shown for C2C12 myotubes (Lee 2004; Stitt et al. 2004). Also, C2C12 myotubes display a hypertrophic response following treatment with insulin-like growth factor-1 (IGF-1; Semsarian et al. 1999). Therefore, to examine the response of primary human skeletal muscle cell cultures to these factors, myotubes derived from HSMM and SkMDC were treated with various concentrations of Dex, Mstn, or IGF-1 for 48 h and then fixed and immuno-stained for MYH2. Nuclei were visualized by Hoechst-staining. Representative images of untreated myotubes or those treated with Mstn (1 μg/ml) or Dex (50 μM) are shown in Fig. 2A and with IGF-1 (1 μg/ml) are shown in Fig. 2B. HSMM and SkMDC undergo an atrophy-like response to both Mstn and Dex treatment, while only HSMM display a hypertrophy-like response to IGF-1. Total myotube areas were measured as described in Semsarian et al. (1999) and Lecomte et al. (2010) and results were plotted as the percent decrease or increase in myotube area compared to untreated control cultures (Fig. 2A, B). The plots show that Mstn and Dex induced statistically significant decreases in myotube area over untreated control cultures at all concentrations (except for the HSMM 250 ng/ml Mstn-treated group which showed decreased myotube area but failed to reach statistical significance; Fig. 2A). Quantification of IGF-1 effects on both cell populations confirmed that only HSMM cultures responded with a hypertrophic response, with significant increases in myotube area at 100 and 1,000 ng/ml IGF-1, (Fig. 2C). Statistical significance, using Student’s t test was performed by comparing the total myotube area in multiple untreated cultures with the total myotube area in treated cultures and then assigning statistical significance (*) if p < 0.05.Figure 2.

Fig2: Induction of atrophy and hypertrophy in HSMM and SkMDC myotubes. (A) Differentiated myotubes were treated with a concentration series of Mstn or Dex for 48 h. Representative images of MYH2/Hoechst-labeled myotubes treated with 1 μg/ml Mstn or 50 μM Dex for 48 h. Plots of the percent decrease, from untreated cells, in myotube area at each concentration of test agent are shown. (All plots represent means ± SEM, n = 15. *p < 0.05 vs. untreated using Student’s t test. All images at ×10 magnification; scale bar = 100 microns). (B) Differentiated myotubes were treated with a concentration series of IGF-1 for 48 h. Representative images of MYH2/Hoechst-labeled myotubes following treatment with 1 μg/ml insulin-like growth factor-1 (IGF-1). Plots show the percent increase, from untreated cultures, in myotube area at each concentration of IGF-1. (All plots represent means ± SEM, n = 15. *p < 0.05 vs. untreated using Student’s t test. All images at ×10 magnification; scale bar = 100 microns). (C) Western blots of myotube lysates from cultures treated with 1 μg/ml Mstn, 50 μM Dex, or 1 μg/ml IGF-1 for 48 h and probed with antibodies to phosphorylated- and total Smad2 and phosphorylated- and total AKT. Anti-alpha tubulin was used as a loading control.

Mentions:
Dexamethasone, a synthetic steroid, and myostatin, a growth factor which is a negative regulator of muscle mass, are well-characterized inducers of myotube atrophy, as shown for C2C12 myotubes (Lee 2004; Stitt et al. 2004). Also, C2C12 myotubes display a hypertrophic response following treatment with insulin-like growth factor-1 (IGF-1; Semsarian et al. 1999). Therefore, to examine the response of primary human skeletal muscle cell cultures to these factors, myotubes derived from HSMM and SkMDC were treated with various concentrations of Dex, Mstn, or IGF-1 for 48 h and then fixed and immuno-stained for MYH2. Nuclei were visualized by Hoechst-staining. Representative images of untreated myotubes or those treated with Mstn (1 μg/ml) or Dex (50 μM) are shown in Fig. 2A and with IGF-1 (1 μg/ml) are shown in Fig. 2B. HSMM and SkMDC undergo an atrophy-like response to both Mstn and Dex treatment, while only HSMM display a hypertrophy-like response to IGF-1. Total myotube areas were measured as described in Semsarian et al. (1999) and Lecomte et al. (2010) and results were plotted as the percent decrease or increase in myotube area compared to untreated control cultures (Fig. 2A, B). The plots show that Mstn and Dex induced statistically significant decreases in myotube area over untreated control cultures at all concentrations (except for the HSMM 250 ng/ml Mstn-treated group which showed decreased myotube area but failed to reach statistical significance; Fig. 2A). Quantification of IGF-1 effects on both cell populations confirmed that only HSMM cultures responded with a hypertrophic response, with significant increases in myotube area at 100 and 1,000 ng/ml IGF-1, (Fig. 2C). Statistical significance, using Student’s t test was performed by comparing the total myotube area in multiple untreated cultures with the total myotube area in treated cultures and then assigning statistical significance (*) if p < 0.05.Figure 2.

Bottom Line:
Primary human skeletal muscle cells have recently become available from a number of commercial vendors.However, only limited characterization of these cells has been reported to date.Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification.

ABSTRACTThere is a significant unmet need for safe, anabolic muscle therapies to treat diseases and conditions associated with severe muscle weakness and frailty. The identification of such therapies requires appropriate cell-based screening assays to select compounds for further development using animal models. Primary human skeletal muscle cells have recently become available from a number of commercial vendors. Such cells may be valuable for studying the mechanisms that direct muscle differentiation, and for identifying and characterizing novel therapeutic approaches for the treatment of age- and injury-induced muscle disorders. However, only limited characterization of these cells has been reported to date. Therefore, we have examined four primary human muscle cell preparations from three different vendors for their capacity to differentiate into multinucleated myotubes. Two of the preparations demonstrated robust myotube formation and expressed characteristic markers of muscle differentiation. Furthermore, these myotubes could be induced to undergo morphological atrophy- and hypertrophy-like responses, and atrophy could be blocked with an inhibitor of myostatin signaling, a pathway that is known to negatively regulate muscle mass. Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification. Taken together, our results indicate that primary human muscle cells can be a useful system for studying muscle differentiation, and may also provide tools for studying new therapeutic molecules for the treatment of muscle disease.